DNA Synthetic Activity Research Articles

Manganese Is a Strong Specific Activator of the RNA Synthetic Activity of Human Polη.

DNA polymerase η (Polη) is a translesion synthesis polymerase that can bypass different DNA lesions with varying efficiency and fidelity. Its most well-known function is the error-free bypass of ultraviolet light-induced cyclobutane pyrimidine dimers. The lack of this unique ability in humans leads to the development of a cancer-predisposing disease, the variant form of xeroderma pigmentosum. Human Polη can insert rNTPs during DNA synthesis, though with much lower efficiency than dNTPs, and it can even extend an RNA chain with ribonucleotides. We have previously shown that Mn2+ is a specific activator of the RNA synthetic activity of yeast Polη that increases the efficiency of the reaction by several thousand-fold over Mg2+. In this study, our goal was to investigate the metal cofactor dependence of RNA synthesis by human Polη. We found that out of the investigated metal cations, only Mn2+ supported robust RNA synthesis. Steady state kinetic analysis showed that Mn2+ activated the reaction a thousand-fold compared to Mg2+, even during DNA damage bypass opposite 8-oxoG and TT dimer. Our results revealed a two order of magnitude higher affinity of human Polη towards ribonucleotides in the presence of Mn2+ compared to Mg2+. It is noteworthy that activation occurred without lowering the base selectivity of the enzyme on undamaged templates, whereas the fidelity decreased across a TT dimer. In summary, our data strongly suggest that, like with its yeast homolog, Mn2+ is the proper metal cofactor of hPolη during RNA chain extension, and selective metal cofactor utilization contributes to switching between its DNA and RNA synthetic activities.

Adenine Radical Cation Formation by a Ligand-Centered Excited State of an Intercalated Chromium Polypyridyl Complex Leads to Enhanced DNA Photo-oxidation.

Assessment of the DNA photo-oxidation and synthetic photocatalytic activity of chromium polypyridyl complexes is dominated by consideration of their long-lived metal-centered excited states. Here we report the participation of the excited states of [Cr(TMP)2dppz]3+ (1) (TMP = 3,4,7,8-tetramethyl-1,10-phenanthroline; dppz = dipyrido[3,2-a:2′,3′-c]phenazine) in DNA photoreactions. The interactions of enantiomers of 1 with natural DNA or with oligodeoxynucleotides with varying AT content (0–100%) have been studied by steady state UV/visible absorption and luminescence spectroscopic methods, and the emission of 1 is found to be quenched in all systems. The time-resolved infrared (TRIR) and visible absorption spectra (TA) of 1 following excitation in the region between 350 to 400 nm reveal the presence of relatively long-lived dppz-centered states which eventually yield the emissive metal-centered state. The dppz-localized states are fully quenched when bound by GC base pairs and partially so in the presence of an AT base-pair system to generate purine radical cations. The sensitized formation of the adenine radical cation species (A•+T) is identified by assigning the TRIR spectra with help of DFT calculations. In natural DNA and oligodeoxynucleotides containing a mixture of AT and GC of base pairs, the observed time-resolved spectra are consistent with eventual photo-oxidation occurring predominantly at guanine through hole migration between base pairs. The combined targeting of purines leads to enhanced photo-oxidation of guanine. These results show that DNA photo-oxidation by the intercalated 1, which locates the dppz in contact with the target purines, is dominated by the LC centered excited state. This work has implications for future phototherapeutics and photocatalysis.

Cytoprotective Effect of Echinochrome A in Primary Culture of Pulmonary Fibroblasts from Albino Rats under Conditions of Oxidative Stress.

We studied the effect of echinochrome A on the primary culture of pulmonary fibroblasts under normal conditions and under oxidative stress. Exposure to echinochrome A (100 μM, 6 h) did not affect the production of superoxide radical by fibroblasts, area of their nuclei, and number of nucleoli, but reduced the total area of nucleolar organizer regions against the background of stable proliferative activity of the culture. Two-hour oxidative stress induced by hydrogen peroxide solution (60 μM) increased the generation of superoxide radical, decreased DNA-synthetic activity of fibroblasts, area of fibroblast nuclei, and total area of nucleolar organizer regions. Preliminary administration of echinochrome A significantly attenuated the damaging effect of oxidative stress: the intensity of production of superoxide radicals decreased, DNA-synthetic activity and nucleus area of fibroblasts partially recovered; normalization of the total area of nucleoli was accompanied by an increase in their number.

The influence of non-opiate analogue of leu-enkephalin to the cardiac consequences of intrauterine hypoxia of albino rats

Objective ― Our study aimed to evaluate the possibility of correcting cardiac consequences of intrauterine hypoxia (IUH) by injecting leu-enkephalin analog, lacking affinity for opiate receptors, in the early postnatal period. Material and Methods ― To model IUH, we placed pregnant Wistar rats in a hypobaric chamber with an oxygen partial pressure of 52 mmHg. The procedure was repeated for 4 h daily over the 15th-19th days of gestation. From the 2nd through the 6th days of their lives, the offspring were injected intraperitoneally with non-opiate leu-enkephalin analog at a dose of 100 μg/kg (NALE: Phe-D-Ala-Gly-Phe-Leu-Arg). This analog did not have affinity for opiate receptors. The 7- and 60-day old offspring of female rats subjected to IUH were investigated. The control group included the descendants of intact animals. We investigated gravimetric indicators, DNA-synthetic activity of cardiomyocytes (CMC) by tritium-labeled thymidine autoradiography method, the size of the CMC nuclei, as well as size and amount of nucleoli in the CMC nuclei. The activity of free radical oxidation was evaluated in cardiac homogenates by chemiluminescence. Results ― In 7-day old rats subjected to IUH vs. control animals, we observed decreases in body mass by 32.6%, in heart mass by 27.3%; in the proportion of 3Н-thymidine labeled CMC nuclei by 32.7% in the left ventricle and by 30.4% in the right ventricle; in the number of nucleoli in the CMC nuclei (in the left ventricle: control – 2.384±0.027, IUH – 2.282±0.027*, p<0.05; in the right ventricle: control – 2.409±0.038; IUH – 2.240±0.012*, p<0, 05). Increase in CML indices of cardiac homogenates was revealed, indicating the activation of free radical oxidation. In 7-day old rats subjected to IUH and administration of the NALE peptide from the 2nd through the 6th days of their lives, the proportion of 3H-thymidine labeled nuclei in the CMC did not differ from the control (in the left ventricle: control – 12.79±0.89%, IUH + NALE – 10.98±0.95%, p>0.05; in the right ventricle: control – 11.61±0.78%; IUH + NALE – 11.26±0.58%, p>0.05). The number of nucleoli in the CMC nuclei of the left and right ventricles in the heart of 7-day old animals in the IUH + NALE group did not differ from the control too. The CML indices of heart homogenates in the IUH + NALE group were significantly lower than those in the IUH group. In 60-day old male rats exposed to IUH, there was a decrease in heart mass by 18.5%, sizes of CMC nuclei by 7.5% and 16.1% in the left and right ventricles, respectively, and in the total nucleoli area in the CMC nuclei of the left ventricle (control – 3.953±0.085; IUH – 3.372±0.078*; p<0.05). In 60-day old male rats subjected to IUH and injections of the NALE peptide from the 2nd to the 6th days of their lives, heart mass (control – 692.73±26.81 mg; IUH + NALE – 631.0±29.79 mg; p>0.05) and the size of the CMC nuclei of the right ventricle (control – 54.25±0.84; IUH + NALE – 55.24±0.94; p>0.05) did not differ significantly from the control. The size of the nuclei, the number and size of the nucleoli in the CMC of the left ventricle, as well as the area of the nucleoli in the CMC of the right ventricle in 60-day old male rats of the IUH + NALE group significantly exceeded control group values. Conclusion ― Administration of the NALE peptide to albino rats subjected to IUH normalized DNA-synthetic activity and the number of nucleoli in the nuclei of CMC in 7-day old animals, and also reduced the severity of oxidative stress in the heart tissue. In 60-day old albino male rats exposed to IUH, injecting NALE from the 2nd to the 6th days of their lives eliminated declines in heart mass and sizes of the CMC nuclei and nucleoli, and also led to an increase in the values of the nucleus-and-nucleolus complex indices compared with the control.

Germination and the Early Stages of Seedling Development in Brachypodium distachyon.

Successful germination and seedling development are crucial steps in the growth of a new plant. In this study, we investigated the course of the cell cycle during germination in relation to grain hydration in the model grass Brachypodium distachyon (Brachypodium) for the first time. Flow cytometry was performed to monitor the cell cycle progression during germination and to estimate DNA content in embryo tissues. The analyses of whole zygotic embryos revealed that the relative DNA content was 2C, 4C, 8C, and 16C. Endoreplicated nuclei were detected in the scutellum and coleorhiza cells, whereas the rest of the embryo tissues only had nuclei with a 2C and 4C DNA content. This study was accompanied by a spatiotemporal profile analysis of the DNA synthetic activity in the organs of Brachypodium embryos during germination using EdU labelling. Upon imbibition, nuclear DNA replication was initiated in the radicle within 11 h and subsequently spread towards the plumule. The first EdU-labelled prophases were observed after 14 h of imbibition. Analysis of selected genes that are involved in the regulation of the cell cycle, such as those encoding cyclin-dependent kinases and cyclins, demonstrated an increase in their expression profiles.

Analysis of Gene Expression Changes in PHA-M Stimulated Lymphocytes – Unraveling PHA Activity as Prerequisite for Dicentric Chromosome Analysis

Dicentric chromosome analysis (DCA) is the gold standard for individual radiation dose assessment. However, DCA is limited by the time-consuming phytohemagglutinin (PHA)-mediated lymphocyte activation. In this study using human peripheral blood lymphocytes, we investigated PHA-associated whole genome gene expression changes to elucidate this process and sought to identify suitable gene targets as a means of meeting our long-term objective of accelerating cell cycle kinetics to reduce DCA culture time. Human peripheral whole blood from three healthy donors was separately cultured in RPMI/FCS/antibiotics with BrdU and PHA-M. Diluted whole blood samples were transferred into PAXgene tubes at 0, 12, 24 and 36 h culture time. RNA was isolated and aliquots were used for whole genome gene expression screening. Microarray results were validated using qRT-PCR and differentially expressed genes [significantly (FDR corrected) twofold different from the 0 h value reference] were analyzed using several bioinformatic tools. The cell cycle positions and DNA-synthetic activities of lymphocytes were determined by analyzing the correlated total DNA content and incorporated BrdU level with flow cytometry after continued BrdU incubation. From 42,545 transcripts of the whole genome microarray 47.6%, on average, appeared expressed. The number of differentially expressed genes increased linearly from 855 to 2,858 and 4,607 at 12, 24 and 36 h after PHA addition, respectively. Approximately 2-3 times more up- than downregulated genes were observed with several hundred genes differentially expressed at each time point. Earliest enrichment was observed for gene sets related to the nucleus (12 h) followed by genes assigned to intracellular structures such as organelles (24 h) and finally genes related to the membrane and the extracellular matrix were enriched (36 h). Early gene expression changes at 12 h, in particular, were associated with protein classes such as chemokines/cytokines (e.g., CXCL1, CXCL2) and chaperones. Genes coding for biological processes involved in cell cycle control (e.g., MYBL2, RBL1, CCNA, CCNE) and DNA replication (e.g., POLA, POLE, MCM) appeared enriched at 24 h and later, but many more biological processes (42 altogether) showed enrichment as well. Flow cytometry data fit together with gene expression and bioinformatic analyses as cell cycle transition into S phase was observed with interindividual differences from 12 h onward, whereas progression into G2 as well as into the second G1 occurred from 36 h onward after activation. Gene set enrichment analysis over time identifies, in particular, two molecular categories of PHA-responsive gene targets (cytokine and cell cycle control genes). Based on that analysis target genes for cell cycle acceleration in lymphocytes have been identified ( CDKN1A/B/C, RBL-1/RBL-2, E2F2, Deaf-1), and it remains undetermined whether the time expenditure for DCA can be reduced by influencing gene expression involved in the regulatory circuits controlling PHA-associated cell cycle entry and/or progression at a specific early cell cycle phase.

Alcoholic extracts of Epilobium, Urtica dioica and Evernia prunastri with 5-fluorouracil in controlling murine colon carcinoma cell growth in vitro

In recent years, there has been growing interest in plant-derived products for their bioactive properties., Results and Conclusion. This study aimed to verify the effect of an herbal-food supplement, comprising alcoholic extracts of Epilobium, Urtica dioica and Evernia prunastri (YATROS, Etnopharma, Turin, Italy), alone or in combination with 5-Fluorouracil, on cell growth, DNA synthesis, cell-cycle profile, cytoskeleton reorganization and E-cadherin protein expression, in the murine colon carcinoma cell line C26. Assays used were respectively the Neutral Red uptake assay, median effect analysis, tritiated thymidine incorporation, flow cytometry, Phalloidin staining, and immunohistochemistry. A chemical fingerprint and multidimensional HPLC-UV-HRMS analysis was also performed on the herbal-food supplements. Intriguingly, a substantial synergistic inhibitory effects on cell growth, DNA synthetic activity and percentage of cells in S phase were noticed when YATROS was combined with 5-Fluorouracil, reducing both dose and timing of exposure of C26 cells to the drug. Moreover, by inducing cytoskeleton reorganization and E-cadherin expression, YATROS can interfere with tumor cell invasiveness. These indications of an agonistic interaction between YATROS and 5-Fluorouracil in colon carcinoma cells may be of interest for clinical applications, and thus merit further investigation.

697 Effects of Gadodiamide on cell proliferation and collagen production in cultured human dermal fibroblasts

Nephrogenic systemic fibrosis (NSF) is a disease characterized by fibrosis of the systemic organs in patients with renal failure. Following the findings of recent epidemiological studies and the finding of gadolinium (Gd) in the skin tissue of NSF patients, it is now definitely known that the use of Gd contrast agents can trigger NSF. To date, however, the exact mechanism underlying the induction of fibrosis in various organs by Gd remains unexplained. The present study was undertaken to evaluate the influence of Gd on the proliferation activity and collagen production of cultured fibroblasts. Normal human dermis-derived fibroblasts were incubated in the presence of gadodiamide (GA). The proliferation activity of the cells was assessed on the basis of the cell counts in the fibroblast growth curve and the DNA-synthetic activity of the cells (indicator; level of 3H-thymidine uptake by cells). The collagen production was evaluated by densitometric measurement of the quantity of collagen through electrophoresis and fluorography after incorporation of 3H-proline into the procollagens. Furthermore, the expression levels of the genes for type I and III collagen were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. The cell count tended to be higher when the fibroblasts were incubated in medium containing GA, furthermore, the DNA synthetic activity also rose in a concentration-dependent manenr in the GA-treated group as compared to that in the control group. No significant changes in either the collagen production or the collagen gene expression levels were noted in cultures containing GA. These results suggest that the formation of sclerosing lesions in patients with NSF may be attributable to the effect of GA of enhancing the growth activity of fibroblasts.

Effects of Gadodiamide on cell proliferation and collagen production in cultured human dermal fibroblasts.

Nephrogenic systemic fibrosis (NSF) is a disease characterized by fibrosis of the systemic organs in patients with renal failure. Following the findings of recent epidemiological studies and the finding of gadolinium (Gd) in the skin tissue of NSF patients, it is now definitely known that the use of Gd contrast agents can trigger NSF. To date, however, the exact mechanism underlying the induction of fibrosis in various organs by Gd remains unexplained. This study was undertaken to evaluate the influence of Gd on the proliferation activity and collagen production of cultured fibroblasts. Normal human dermis-derived fibroblasts were incubated in the presence of gadodiamide (GA) in the concentration range of 5×10-7 to 5×10-3 M. The proliferation activity of the cells was assessed on the basis of the cell counts in the fibroblast growth curve and the DNA-synthetic activity of the cells (indicator; level of 3H-thymidine uptake by cells). The collagen production was evaluated by densitometric measurement of the quantity of collagen through electrophoresis and fluorography after incorporation of 3H-proline into the procollagens. Furthermore, the expression levels of the genes for type I and III collagen were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. The cell count tended to be higher when the fibroblasts were incubated in medium containing GA in the concentration range of 5×10-7 to 5×10-4M as compared to that in the GA-free control cultures; furthermore, the DNA-synthetic activity also rose in a concentration-dependent manner in the GA-treated group as compared to that in the control group. No significant changes in either the collagen production or the collagen gene expression levels were noted in cultures containing GA at concentrations between 5×10-7 and 5×10-3 M. These results suggest that the formation of sclerosing lesions in patients with NSF may be attributable to the effect of GA of enhancing the growth activity of fibroblasts.

Effects of antenatal hypoxia on tissue homeostasis in the myocardium of albino rats: early and delayed consequences.

Body weight, absolute and relative heart weight, DNA-synthetic activity of cardiomyocytes, and number of nucleoli in cardiomyocyte nuclei were reduced in newborn albino rats exposed to antennal hypoxia. All these changes developed against the background of oxidative stress. In mature animals, the absolute and relative heart weights were decreased, but the ratio of mononuclear cardiomyocytes in both ventricles was higher than in the control. These changes were accompanied by an increase in the percent of collagen fibers in the myocardial stroma. In the right ventricle, an increase in the cardiomyocyte length to width ratio and a decrease in the number of nucleoli were found. The observed changes can probably induce heart function disturbances under pathological conditions.

Effects of antenatal hypoxia on the parameters of tissue homeostasis in the liver of albino rats.

We studied age-dependent dynamics of some liver parameters in albino rats after antenatal hypoxia. A decrease in body weight, absolute liver weight in newborn and mature rats, and a reduction of relative weight of the liver in mature animals were observed under these conditions. It was found that antenatal hypoxia promotes a decrease in DNA-synthetic activity of hepatocytes, intensifies free radical processes in the liver of newborn rats, and reduces the number of nucleoli in hepatocytes. In mature animals, antenatal hypoxia was followed by a decrease in hepatocyte sizes and area of nucleoli, an increase in the number of binuclear cells relative to mononuclear cells, and a decrease in the number of nucleoli in hepatocyte nuclei. These data can reflect impairment of tissue homeostasis in the liver of mammals exposed to antenatal hypoxia.

Low-dose steroid pretreatment ameliorates the transient impairment of liver regeneration

To determine if liver regeneration (LR) could be disturbed following radiofrequency (RF) ablation and whether modification of LR by steroid administration occurs. Sham operation, partial hepatectomy (PH), and partial hepatectomy with radiofrequency ablation (PHA) were performed on adult Fisher 344 rats. We investigated the recovery of liver volume, DNA synthetic activities, serum cytokine/chemokine levels and signal transducers and activators of transcription 3 DNA-binding activities in the nucleus after the operations. Additionally, the effects of steroid (dexamethasone) pretreatment in the PH group (S-PH) and the PHA group (S-PHA) were compared. The LR after PHA was impaired, with high serum cytokine/chemokine induction compared to PH, although the ratio of the residual liver weight to body weight was not significantly different. Steroid pretreatment disturbed LR in the S-PH group. On the other hand, low-dose steroid pretreatment improved LR and suppressed tumor necrosis factor (TNF)-α elevation in the S-PHA group, with recovery of STAT3 DNA-binding activity. On the other hand, low-dose steroid pretreatment improved LR and suppressed TNF-α elevation in the S-PHA group, with recovery of STAT3 DNA-binding activity. LR is disturbed after RF ablation, with high serum cytokine/chemokine induction. Low-dose steroid administration can improve LR after RF ablation with TNF-α suppression.

Effects of Analogues of Hydra Peptide Morphogen on DNA Synthesis in the Myocardium of Newborn Albino Rats

DNA-synthetic activity of myocardial cells was studied by (3)H-thymidine autoradiography in newborn albino rats after intraperitoneal injection of hydra peptide morphogen and its analogues. Administration of hydra peptide morphogen stimulated proliferative activity in the myocardium. Short analogues of hydra peptide morphogen, 6C and 3C peptides, produced a similar effect. Administration of arginine-containing analogue of hydra peptide morphogen significantly reduced the number of DNA-synthesizing nuclei in the ventricular myocardium of newborn albino rats. The role of the structure of the peptide molecule in the realization of the morphogenetic effects of hydra peptide morphogen is discussed.

Betacellulin-Induced Beta Cell Proliferation and Regeneration Is Mediated by Activation of ErbB-1 and ErbB-2 Receptors

BackgroundBetacellulin (BTC), a member of the epidermal growth factor family, is known to play an important role in regulating growth and differentiation of pancreatic beta cells. Growth-promoting actions of BTC are mediated by epidermal growth factor receptors (ErbBs), namely ErbB-1, ErbB-2, ErbB-3 and ErbB-4; however, the exact mechanism for beta cell proliferation has not been elucidated. Therefore, we investigated which ErbBs are involved and some molecular mechanisms by which BTC regulates beta cell proliferation.Methodology/Principal FindingsThe expression of ErbB-1, ErbB-2, ErbB-3, and ErbB-4 mRNA was detected by RT-PCR in both a beta cell line (MIN-6 cells) and C57BL/6 mouse islets. Immunoprecipitation and western blotting analysis showed that BTC treatment of MIN-6 cells induced phosphorylation of only ErbB-1 and ErbB-2 among the four EGF receptors. BTC treatment resulted in DNA synthetic activity, cell cycle progression, and bromodeoxyuridine (BrdU)-positive staining. The proliferative effect was blocked by treatment with AG1478 or AG825, specific tyrosine kinase inhibitors of ErbB-1 and ErbB-2, respectively. BTC treatment increased mRNA and protein levels of insulin receptor substrate-2 (IRS-2), and this was blocked by the ErbB-1 and ErbB-2 inhibitors. Inhibition of IRS-2 by siRNA blocked cell cycle progression induced by BTC treatment. Streptozotocin-induced diabetic mice injected with a recombinant adenovirus expressing BTC and treated with AG1478 or AG825 showed reduced islet size, reduced numbers of BrdU-positive cells in the islets, and did not attain BTC-mediated remission of diabetes.Conclusions/SignificanceThese results suggest that BTC exerts proliferative activity on beta cells through the activation of ErbB-1 and ErbB-2 receptors, which may increase IRS-2 expression, contributing to the regeneration of beta cells.

The Block of DNA Polymerase δ Strand Displacement Activity by an Abasic Site Can Be Rescued by the Concerted Action of DNA Polymerase β and Flap Endonuclease 1

Abasic (AP) sites are very frequent and dangerous DNA lesions. Their ability to block the advancement of a replication fork has been always viewed as a consequence of their inhibitory effect on the DNA synthetic activity of replicative DNA polymerases (DNA pols). Here we show that AP sites can also affect the strand displacement activity of the lagging strand DNA pol delta, thus preventing proper Okazaki fragment maturation. This block can be overcome through a polymerase switch, involving the combined physical and functional interaction of DNA pol beta and Flap endonuclease 1. Our data identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism and suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling.

Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC 50 of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases α, δ and ɛ is 15, 45 and 125 μM, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC 50 = 88 μM), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2–13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr(III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication correlates with the genotoxic and cytotoxic effects of this metal ion; and promotes cell killing via inhibition of the DNA polymerase activity mediating the DNA replication and repair processes utilized by human cells.

PIK3IP1, a Negative Regulator of PI3K, Suppresses the Development of Hepatocellular Carcinoma

Phosphatidylinositol-3-kinase (PI3K) is a well-known regulator of cell division, motility, and survival in most cell types. Recently, we characterized a novel protein that we call PI3K Interacting Protein 1 (PIK3IP1), which binds to the p110 catalytic subunit of PI3K and reduces its activity in vitro. Little is known about the role of PIK3IP1 in normal and neoplastic growth in vivo. Proper liver function and development depend on intact PI3K signal transduction; when dysregulated, the PI3K pathway is linked to the development of liver cancer. To begin to dissect the contribution of PIK3IP1 to hepatic PI3K signaling in vivo and to liver tumorigenesis in particular, we formulated the following hypothesis: because PIK3IP1 down-regulates PI3K signaling and uncontrolled PI3K signaling is associated with liver cancer, then PIK3IP1-mediated down-regulation of the PI3K pathway should inhibit hepatocellular carcinoma (HCC) development. To test this idea, we generated transgenic mice overexpressing PIK3IP1 in hepatocytes in a mouse strain prone to develop HCC. Isolated PIK3IP1 transgenic mouse hepatocytes showed blunted PI3K signaling, DNA synthetic activity, motility, and survival compared with controls. In vivo, spontaneous liver tumorigenesis was significantly dampened in the transgenic animals. This was accompanied by decreased hepatic PI3K activity and reduced hepatocyte proliferation in the transgenics compared with controls. We also observed that human HCC expressed less PIK3IP1 protein than adjacent matched liver tissue. Our data show that PIK3IP1 is an important regulator of PI3K in vivo, and its dysregulation can contribute to liver carcinogenesis.

Biochemical properties of a plastidial DNA polymerase of rice

Plastids are organelles unique to plant cells and are responsible for photosynthesis and other metabolic functions. Despite their important cellular roles, relatively little is known about the mechanism of plastidial DNA replication and repair. Recently, we identified a novel DNA polymerase in Oryza Sativa L. (OsPOLP1, formerly termed OsPolI-like) that is homologous to prokaryotic DNA polymerase Is (PolIs), and suggested that this polymerase might be involved in plastidial DNA replication and repair. Here, we propose to rename the plant PolI homologs as DNA polymerase pi (POLP), and investigate the biochemical properties of full-length OsPOLP1. The purified OsPOLP1 elongated both DNA and RNA primer hybridized to a DNA template, and possessed a 3' exonuclease activity. Moreover, OsPOLP1 displayed high processivity and fidelity, indicating that this polymerase has the biochemical characteristics appropriate for DNA replication. We found that POLPs have two extra sequences in the polymerase domain that are absent in prokaryotic PolIs. Deletion of either insert from OsPOLP1 caused a decrease in DNA synthetic activity, processivity, and DNA binding activity. In addition, OsPOLP1 efficiently catalyzed strand displacement on nicked DNA with a 5'-deoxyribose phosphate, suggesting that this enzyme might be involved in a repair pathway similar to long-patch base excision repair. These results provide insights into the possible role of POLPs in plastidial DNA replication and repair.

Spatial Patterns of DNA Replication, Protein Synthesis, and Oxygen Concentration within Bacterial Biofilms Reveal Diverse Physiological States

It has long been suspected that microbial biofilms harbor cells in a variety of activity states, but there have been few direct experimental visualizations of this physiological heterogeneity. Spatial patterns of DNA replication and protein synthetic activity were imaged and quantified in staphylococcal biofilms using immunofluorescent detection of pulse-labeled DNA and also an inducible green fluorescent protein (GFP) construct. Stratified patterns of DNA synthetic and protein synthetic activity were observed in all three biofilm systems to which the techniques were applied. In a colony biofilm system, the dimensions of the zone of anabolism at the air interface ranged from 16 to 38 microm and corresponded with the depth of oxygen penetration measured with a microelectrode. A second zone of activity was observed along the nutrient interface of the biofilm. Much of the biofilm was anabolically inactive. Since dead cells constituted only 10% of the biofilm population, most of the inactive cells in the biofilm were still viable. Collectively, these results suggest that staphylococcal biofilms contain cells in at least four distinct states: growing aerobically, growing fermentatively, dead, and dormant. The variety of activity states represented in a biofilm may contribute to the special ecology and tolerance to antimicrobial agents of biofilms.

PIK3IP1, a novel negative regulator of PI3K, inhibits hepatocyte DNA synthesis, migration and survival in vitro

PI3K Interacting Protein 1 (PIK3IP1) is a transmembrane protein we discovered which binds to the p110 catalytic subunit of PI3K and inhibits PI3K, a well-known regulator of cell proliferation, migration and survival in most cell types. We have determined that the endogenous expression of PIK3IP1 in hepatocytes is low at the mRNA and protein levels inversely correlating with high PI3K activity in this cell type. Based on our findings, we hypothesized that overexpression of PIK3IP1 in hepatocytes could alter the natural activity of the PI3K pathway in this cell type. To test this hypothesis, we developed a PIK3IP1 transgenic mouse model directing expression of PIK3IP1 to hepatocytes under the regulatory control of the albumin promoter. Analysis of the livers of the transgenic animals showed decreased PI3K activity and reduced hepatocyte proliferation as compared to controls. Next, we isolated hepatocytes from the mice and observed a reduction in PI3K signaling in the cultured transgenic hepatocytes overexpressing PIK3IP1 as compared to non-transgenic littermate controls. We next examined the cultured hepatocytes for their DNA synthetic activity, migration and motility as well as resistance to apoptosis in a variety of assays. We detected a lower DNA synthetic rate as well as reduced motility and migration in the PIK3IP1 transgenic hepatocytes as compared to controls. Furthermore, PIK3IP1 overexpression enhanced UV induced apoptosis, as confirmed by both TUNEL staining and cleaved caspase-3 expression. In conclusion, our data suggest that overexpression of PIK3IP1 suppresses hepatocyte DNA synthesis, migration and survival through inhibition of the PI3K pathway.